High-Throughput Sequencing Strategy for Microsatellite Genotyping Using Neotropical Fish as a Model

Abstract

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple <i>loci</i> from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite <i>loci</i> for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite <i>loci</i> from the fish <i>Prochilodus costatus</i>, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per <i>locus</i> when genotyping eight <i>loci</i>. Assuming a minimum requirement of 10 reads per <i>loci</i>, two to four times more <i>loci</i> could be tested in each run, depending on the quality of the PCR reaction of each <i>locus</i>. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.